Ion-exchange chromatography is a frequently used chromatography technique that separates ions and polar molecules based on their charge. It can be used for almost any type of charged molecule including large proteins, polypeptides, nucleic acids, polynucleotides, small nucleotides and amino acids. The surface of the stationary phase displays ionic functional groups, which interact with analyte ions of opposite charge through columbic (ionic) interactions. Ion-exchange chromatography is thus divided into cation-exchange chromatography and anion-exchange chromatography. In cation-exchange chromatography, positively charged cations are retained because the stationary phase displays a negatively charged functional group, whereas in anion-exchange chromatography, anions are retained by positively charged functional groups on the stationary phase.
Biomolecules are any molecule that are produced by a living organism, including large macromolecules such as nucleic acids, proteins, polysaccharides and lipids, as well as small molecules such as metabolites and natural products. The ability to efficiently and accurately analyze biomolecules is central to the life science, materials and other industries, for example in drug R&D, medical diagnosis, forensic analysis, genetic and food testing. Ion-exchange chromatography is a key technique for analysis and purification, isolation of various types of biomolecules. For example, anion-exchange high-performance liquid chromatography (HPLC) is often an ideal chromatographic mode for oligonucleotide separations. The counter-ionic interaction of the negatively charged analytes and a stationary phase with a surface which displays positively charged functional groups provides for excellent column retention. With optimized analytical parameters, single nucleotide variants (e.g., N−1, N+1) are often chromatographically specified in the presence of longer (˜30-40 nt) oligonucleotide sequences.
The separation mode is also applied to oligonucleotides that are conjugated to large (˜40K Da) polyethylene-glycol (PEG) moieties. When these molecules are separated under similar analytical conditions, differences in the appearance of the chromatography are noted relative to that observed for the non-PEGylated analogs. Reduced retention times and band broadening in the chromatographic trace are typically found with the PEGylated species. Additionally, impurity peaks are observed with valleys and inflection points presenting with a more ‘vague’ appearance. Single nucleotide variant selectivity may still be observed with such PEGylated analytes with careful choice of analytical parameters, although the peak resolution of the respective species is often observed to be significantly less. The reduced resolution, sensitivity and recovery could substantially impact analytical and preparative functionalities of ion-exchange chromatography as an effective tool for biomolecules.
Previous attempts at addressing the low recovery of impurities involved adding additional organic solvent (e.g., acetonitrile, isopropanol) to the mobile phase regime and also to perform the analysis at higher temperatures. These measures, however, have been largely ineffective.
Thus, there remains an unmet need for novel approaches that effectively address the issues associated with reduced resolution, sensitivity and recovery and to make ion-exchange HPLC an effective analytical and preparative tool for certain analytes.